Process of producing plastic plates with wells by placing a film on a mold and applying hot gas

ABSTRACT

A process serves for producing a plate comprising at least one well havingts opening facing upwards, for receiving chemical and/or biochemical and/or microbiological substances. The process, wherein the wells are made in the plate with the aid of a molding die, comprises the steps of: 
     a) Applying a plastic film, which is deformable by the action of heat, on a molding block which serves as a molding die and which has the negative shape of the wells to be formed; and 
     b) subjecting the plastic film, for a predetermined period of time, to the action of a hot gas flow of a predetermined first temperature, the gas flow impinging upon at least an area of the plastic film covering one of the recessed portions, heating up this area and pressing it into the recessed portion so that it comes to lie flat against the latter&#39;s inner wall which is smooth throughout.

The present invention relates to a process for producing a platecomprising at least one well having its opening facing upwards, forreceiving chemical and/or biochemical and/or microbiological substances,the wells being made in the plate with the aid of a molding die.

The invention also relates to a plate produced in accordance with theprocess.

Plates of this type are marketed by several suppliers and are known asmicrotest plates or microtiter plates.

The known plates consist of polystyrene or polyvinyl chloride and have arigid bottom which is surrounded on all sides by an upwardly projectingedge. Wells arranged in rows and columns are recessed into the bottomfrom above, the bottom having a thickness of more than 1 mm. The volumeof the wells is normally between some hundred microliters and somemilliliters.

It has been known before to cover the edge of the plate with a lid inorder to protect the wells and the interior space bounded by the edgeagainst the ingress of contamination of any type.

The plates are used to keep solutions or substances at constanttemperature. This is effected either for storage purposes, for examplein a refrigerator, or in order to allow a reaction to proceed at aspecific temperature. For the latter purpose, the plates are placed inan incubator which as a rule is set at 37° C.

However, the known plates are not suited for many of the modernchemical, biochemical or microbiological methods. These methods requirethat the reaction solutions be brought to different temperatures as thetest proceeds, which temperatures may vary from far below 0° C. to above110° C. The reactions often have to follow a specific temperatureprofile cycle, which can consist of several heating and/or coolingoperations. The yield and efficiency of the reactions depends in thiscase substantially on the speed at which the temperature change can bebrought about in the different solutions used. Especially in the case ofenzymatic processes in connection with examinations on nucleic acids,rapid temperature changes are required between high temperatures formelting double strands and low temperatures for initiating the reaction.

Some of these new methods have developed to standard procedures where alarge number of samples is subjected continuously and repeatedly to thesame procedural sequence. For aids tests, for example, a large number ofblood samples is examined simultaneously in order to make available manyresults as quickly as possible.

Although the known plates are commercially available with up to 96wells, this number is far too small for the required huge number oftests to be carried out. In addition, the known plates are unsuited forfrequent rapid temperature changes, the processes of cooling them downin a refrigerator and/or heating them up in an incubator taking far toomuch time.

Although it is known to control the temperature of the plates via theirplane underside, the temperature change of the samples, requiring up toseveral minutes, takes far too much time in this case, too.

In addition, conically tapered plastic reaction vessels which have asnap-on lid or a screw lid are known. The known plastic reaction vesselsare, however, as a rule several centimeters high and have an externaldiameter in the range of 10 to 18 mm. The temperature of the solutionscontained therein is changed by controlling the temperature of thereaction vessels as such, via their outsides. This is effected, forexample, by immersing the reaction vessels in water baths. However, ithas also been known to provide temperature controlled metal blocks withbores for the reaction vessels which are then temperature-controlled bytheir contact with the walls of such bores. The heat transfer may beimproved in this case by filling the bores with water or oil.

The temperature change of the samples can be effected at the quickest byintroducing the reaction vessels into different water baths and/or metalblocks which are adjusted to the new desirable temperature. Due to thethickness of the walls of the plastic reaction vessels, however, thesolutions adapt to the new temperature only slowly.

Consequently, the known reaction vessels are not suited for methodsrequiring quick temperature changes. Moreover, the space required by theknown plastic reaction vessels is considerable, if a large number ofsamples is to be processed. And in addition it is a disadvantage that agreat number of lids has to be opened and then closed again, at leastduring preparation of the test, which is rather time-consuming.

It has also been known, in connection with a deep-drawing process, toheat up a rigid plastic plate, to bring it into contact with a moldingdie and to adapt it to the negative or positive form of the molding dieby the application of overpressure or a partial vacuum. However, thisprocess is not suited for producing wells of small volume as the thinplastic films to be used in this case tend to tear rapidly and inaddition no smooth surfaces can be obtained in the small wells. Thisalso has a negative influence on the speed of the temperature change.

Now, it is the object of the present invention to improve the knownmethod in such a way that a plate of the type described above can beproduced which overcomes the described disadvantages. In particular, itshould be possible to produce a plate of this type which, whilst beingsimple to handle, enables quick temperature changes to be effected on alarge number of reaction solutions, also in small volumes.

This object is achieved according to the invention by the fact that theprocess comprises the steps of

a) applying a plastic film, which is deformable by the action of heat,on a molding block which serves as a molding die and which has thenegative shape of the wells to be formed; and

b) subjecting the plastic film, for a predetermined period of time, tothe action of a hot gas flow of a predetermined first temperature, thegas flow impinging upon at least an area of the plastic film coveringone of the recessed portions, heating up this area and pressing it intothe recessed portion so that it comes to lie flat against the latter'sinner wall which is smooth throughout.

This solves the object of the invention in full, the novel process beingcapable of producing perfectly shaped wells displaying a bulged portionprojecting downwards below the film and having a large heat-exchangingsurface. The wall thickness of the wells is thinner than the originalthickness of the plastic film. Due to the small wall thickness and thelarge heat-exchanging surface, rapid heat exchange is guaranteed throughthe walls of the wells. When the heat-exchanging surface of the wells isbrought into contact with a heating or cooling substance having atemperature different than that of the solutions contained in the wells,the latter will assume the new temperature within the time of a fewseconds.

In addition, it is now possible, with the aid of the novel process, toproduce a great number of wells in a rigid, but thin plastic film. Asingle plate may now contain up to thousand wells the temperature ofwhich can be controlled simultaneously via their undersides--for examplewith the aid of a water bath. The resulting possibility to process alarge number of samples in parallel clearly increases the economicefficiency of the particular method used, for example the chemicalmethod.

According to a preferred further development of this novel process, themolding block is maintained at a predetermined second temperature whichis lower than the predetermined first temperature, but above roomtemperature.

This feature provides the surprising advantage that the outsides of thewells become very smooth due to the fact that the air escapes duringproduction without the formation of bubbles. These smooth outer wallscan be brought into engagement, without any problem, with holes ofmatching shape, which may be provided for example in metal blocks,without even the thinnest air layers disturbing the heat transferbetween the metal block and the wall. It is thus possible to achievevery rapid changes in temperature of the plates produced with the aid ofthe novel method, using metal-block thermostats. In addition, the metalblocks give the plates placed thereon good mechanical stability.

Another advantage is achieved with this process by the fact that the hotgas flow issues from a nozzle whose orifice is maintained at a fixeddistance from the plastic film during the fixed period of time.

This enables even very thin plastic films to be processed with the aidof the novel method without the trapped air obstructing the well-moldingprocess. The plates so produced have very thin walls which improves theheat transfer once more.

Furthermore, it is advantageous if the wells, which display a circularcross-section, have a volume smaller than 200 microliters and a diametersmaller than 10 mm, if the orifice of the nozzle has a diameterapproximately equal to the diameter of the wells and if the fixeddistance corresponds approximately to the diameter of the wells.

This feature provides the advantage that smooth outer surfaces andreproducible wells can be produced even for very small volumes.

Furthermore, it is advantageous if the plastic film is a polycarbonatefilm having a thickness smaller than 0.5 mm, if the predetermined firsttemperature is between 250° C. and 300° C., and if the predeterminedsecond temperature is between 90° C. and 110° C.

As a result of these measures, the novel process is capable of producingwells with wall thicknesses below 0.1 mm so that very efficient heattransfer is guaranteed. Compared with the known plates and the knownplastic reaction vessels, the plates produced according to the novelprocess require clearly less space, whilst the number of wells isconsiderably increased. In addition, the quantity of material requiredfor producing the new plates is notably reduced, so that the plates canbe designed as low-cost disposable items.

In the case of a plate produced according to the novel process, the wellhas a heat transfer coefficient which is greater than 5×10⁻⁴ W/(K mm³)and which is defined by the formula W=(A·λ)/(V·x), wherein A is the sizeof the heat-exchanging surface, λ is the thermal conductivity of thematerial forming the wall, V is the volume of the well, x is the wallthickness of the well, measured as the distance between theheat-exchanging surface and the inner surface, and W is theheat-transfer coefficient.

Such a heat transfer coefficient permits quick heat transfer through thewall, into and out of the well.

Further advantages are apparent from the description and the appendeddrawing.

It is obvious that the features which have been mentioned above andwhich are still to be explained below can be used not only in theparticular combination indicated but also in other combinations oralone, without departing from the scope of the present invention.

One illustrative embodiment of the invention is shown in the drawing andis explained in more detail in the following description. In thedrawing:

FIG. 1 shows the plate according to the invention with the wells opentowards the top, in section and in a perspective view;

FIG. 2 shows the plate from FIG. 1, in a sectional view along the lineII--II from FIG. 1;

FIG. 3 shows a process for the production of the plate from FIG. 1, in adiagrammatic representation;

FIG. 4 shows the covering of a plate according to FIG. 1 with a coverfilm, in section and in a perspective view;

FIG. 5 shows the covered plate from FIG. 4 with circular joining seamswhich are positioned around the wells and join the cover film to theplate, as seen in the direction of the arrow V from FIG. 4;

FIG. 6 shows a welding die for producing the joining seams from FIG. 5,in a sectional partial view;

FIG. 7 shows the welding die from FIG. 6, in a partial view from abovealong the arrow VII from FIG. 6;

FIG. 8 shows the joining seam from FIG. 5, in a sectional side viewalong the line VIII--VIII from FIG. 5;

FIG. 9 shows an installation for welding the cover plate from FIG. 4, inwhich several of the welding dies from FIG. 6 are used, in a perspectiveview; and

FIG. 10 shows the use of the welded plate from FIG. 5 in combinationwith a thermoblock in a perspective view and in section.

In FIG. 1 a rectangular flexible plate 2 is shown in section with one ofits longitudinal edges 3 and one of its side edges 4. The plate 2, whichmay for example be made of a rigid plastic film, has a plane upper side5 and an underside 6 parallel thereto. Its thickness, measured betweenthe upper side 5 and the underside 6 is indicated at 7. As can be seenin FIG. 1 , the thickness 7 is small compared with the transversedimensions of the plate 2.

Through-holes 9 are provided and wells 11, which are open towards thetop, are formed in the plate 2. The wells 11 are arranged in rows 12 andcolumns 13, the rows 12 running parallel to the longitudinal edge 3 andthe columns 13 parallel to the side edge 4. The rows 12 and columns 13each have a mutual row spacing or column spacing, indicated at 14 and 15respectively. In the illustrative embodiment shown, the wells 11, whichare of circular cross section, have an internal diameter 16, which canbe better discerned in FIG. 2. The row spacings 14 and the columnspacings 15 are identical, the internal diameter 16 of the wells 11 ofcourse being smaller than the row spacing 14 or the column spacing 15.

The wells 11 lie with their openings 18, which are surrounded by arounded opening edge 17, in the plane of the upper side 5 of the plate2. They have a wall 20 which delimits their interior space 19 and isconstructed in the manner of a beaker-like protuberance 21 and for eachof the wells 11, which are identical to one another, lies below theunderside 6 of the plate 2.

In the remainder of the description "upwards" will be used to indicatethe direction out of the interior space 19 of the wells 11 through theopening 18 and "downwards" accordingly will be used to indicate theopposite direction.

As can be better discerned in FIG. 2, the protuberance 21 has a hollowcylindrical upper section 22 and a hemispherical lower section 23integral therewith, the curved bottom wall 24 of said lower sectionclosing off the well 11 at the bottom. The upper side 5 merges directlyas inner surface 15 into the interior space 19 of the well forming atthe transition the peripheral rounded edge 17, while the underside 6runs, as the outside 28 of the protuberance 21, essentially parallel tothe curved inner surface 25, forming a neck groove 27 surrounding theprotuberance 21. Bridges 29, which separate the individual openings 18from one another, run between the individual wells 11.

As can further be seen in FIG. 2, the bottom wall 24 has the thickness,indicated at 31, which is measured between the inner surface 25 and theoutside 28. In the region of the hollow cylindrical upper section 22, acorrespondingly measured thickness, which is approximately equal to thethickness 31, is indicated at 32. The wells 11 each have a volume 33,which is essentially determined by their depth, indicated at 34, and theinternal diameter 16. The depth 34 is measured between the bottom wall24 and an imaginary maximum filling height indicated at 35 by a dashedline. The filling height 35 is approximately at the height at which thecurved opening edge 17 merges into the vertical inner surface 25.Because of the surface tension and the precurvature associatedtherewith, the fill volume of the substances to be received will besmaller than the maximum volume 33, especially when the volume 33 issmall.

The wells 11 of the plate 2 described thus far serve to receive chemicaland/or biochemical and/or microbiological substances, which are storedor subjected to a reaction in the wells 11. The volume 33 and the numberof wells 11 per plate 2 depend on the substances to be received in thewells 11. In addition to the internal diameter 16 and the depth 34, therow spacing 14 and the column spacing 15 are also largely determined bythe volume 33. The thickness 7 of the plate 2 in the region of thebridges 29 is so chosen that the plate 2 has an adequate strengthdespite the closely adjacent wells 11 and does not break throughbuckling when being transported with filled wells 11. The thicknesses 31and 32 of the wall 20 of the wells 11 are so chosen from the mechanicalstandpoint that the filled wells 11 do not start to tear or even tearoff under the weight of the substances received therein.

In addition to the purely mechanical standpoint, the material from whichthe plate 2 is made and the thicknesses 31 and 32 of the wall 20 arealso chosen from physical standpoints. The thicknesses 31 and 32,which--as can be seen in FIG. 2--are considerably less than thethickness 7, enable good transport of heat into the interior space 19 ofthe wells 11 and out of the interior space 19. By this means it ispossible to cool or to change the temperature of the substances in thewells very rapidly, as the entire outside 28 is brought into contact, asa heat exchange surface 28', with a temperature-control material whichis at the particular temperature desired.

In the illustrative embodiment chosen, the plate 2 is made ofpolycarbonate and has a thermal conductivity of λ=0.21 W per Kelvin andper meter. The thickness 7 is about 0.27 mm and for the thickness 31:x=0.04 mm. The spacings 14 and 15 between the rows 12 and, respectively,the columns 13 are about 10 mm and the volume 33 of the wells 11 is V=85μl. The size of the heat exchange surface 28' corresponds to the outside28 and is: A=75 mm². In accordance with the equation ##EQU1## a heattransfer coefficient of about 4.5×10⁻³ W/(K mm³) results using thesefigures.

It has been found that for such a heat transfer coefficient the heatexchange through the wall 20 takes place so rapidly that the determiningtime factor is the heat conduction in the substances themselves.

Thus, the novel plate 2 enables, for example, a large number ofreactions to be carried out in separate wells 11 within a small space,it being possible to achieve very good thermal control of the reactionsthrough the wall 20 of the wells.

Moreover, the material of the plate 2 is so chosen that opticalanalytical procedures, such as, for example, absorption measurements orfluorescence measurements, are possible through the wall 20 of the wells11. For this purpose, the material must be transparent in the lightwavelength range of interest, i.e it must display neither significantabsorption nor fluorescence emission in this wavelength range.

With reference to FIG. 3, a process for the production of the plate 2from FIG. 1 will now be described. The starting material is a thin film36, for example made of a polycarbonate, which has a thickness 7. Thisfilm 36 is placed on a temperature-controlled shaping block 37, in whichblind holes 38, open towards the top, are provided, which holes, likethe wells 11, are arranged in rows 12 and columns 13. The blind holes 38have a wall surface 40 which surrounds their interior 39 and is smoothand coherent. The dimensions of the blind holes 38 are so chosen thatthey correspond to the external dimensions of the protuberances 21 to beformed: in the chosen example the blind holes have a diameter of about 6mm and a depth of about 4 mm.

A heater, indicated diagrammatically at 43, is provided in the shapingblock 37, which is made of metal, for example aluminium, the shapingblock 37 being heated uniformly to 100° C. by said heater. An air nozzle45, which is moveable in the direction of the arrow 46, is arrangedabove the shaping block 37 in the direction of the blind holes 38. Thedirection 46 is parallel to the columns 13 or the rows 12, so that theair nozzle 45 can be positioned centrally above each individual blindhole 38. The direction 46 is also aligned parallel to the upper side 5of the film 36 placed on the shaping block 37, so that the spacingbetween the air nozzle and the upper side 5 remains constant.

The air nozzle 45 releases a jet 47 of hot air which is at about 280° C.and issues downwards at a velocity of about 2-5 m/sec from its outletorifice 48 approximately vertically to the shaping block 37. The outletorifice 48 has a diameter of about 5 mm and is 4 mm above the upper side5 of the film 36. The air nozzle 45 is positioned successively centrallyabove the individual blind holes 38, where it remains stationary forabout 3 to 5 seconds. By means of the jet 47 of hot air impinging on theupper side 5, the film 36 is heated to such an extent that it isplastically deformable.

The jet 47 of hot air then blows that area of the film 36 originallylocated above the blind hole 38 into the interior 39 of the particularblind hole, this area gradually stretching and the original thickness 7of the film 36 becoming ever smaller in this area until, finally, thewall 20 of the well 11 formed has the thicknesses 31 and 32 indicated inFIG. 2.

In FIG. 3 the righthand well 11/1 has already been completely formed andthe air nozzle 45 is located above the blind hole 38/2, in which thewell 11/2 is just being formed. The bottom 24 of the well 11/2 hasalready partly moved into the interior 39/2 of the blind hole 38/2 andsubsequently will lie with its entire surface against the smooth innerwall of the blind hole 38/2. As can be seen in FIG. 3, the bridge 29,which has the original thickness 7 of the film 36, remains between thewells 11/1 and 11/2. When forming the wells 11, the occluded air escapeswithout the formation of bubbles.

Of course, it is possible to use, instead of one air nozzle 45, severalparallel air nozzles 45, the outlet orifices 48 of which are arranged inthe grid pattern of the columns 13 or the rows 12. In this way,depending on the number of air nozzles 45, all the wells 11 of one row12 or also of one column 13 can be produced at the same time.

As has already been described above, the film 36 consists of apolycarbonate having a thickness of 0.27 mm. Before forming the wells11, the film 36 has a milky turbidity. However, it has been found thatwhen the temperature of the jet 47 of hot air is 280° C. and thetemperature of the shaping block 37 is 100° C. the film 36 becomestransparent in the region of the wall 20 of the completely formed wells11, as is required for the above-mentioned optical analytical methods.Bringing the shaping block 37 to a controlled temperature of 100° C.,which is not necessary for the actual forming of the wells 11, has theadditional effect that the outside 28 of the well 11 lies fully againstthe wall surface 40 of the particular blind hole 38. By this means it isachieved that the outside 28 of each individual well 11 also has asmooth and uniform surface, which is of great advantage for changing thetemperature of substances placed in the wells 11. The reason is that theprotuberances 21 have virtually identical contours, so that they can bebrought, with their heat exchange surface 28', into direct contact withcorrespondingly shaped counter-surfaces in the blind hole bores 38without any layers of air interfering with the heat transfer. This isalso described further below with reference to FIG. 10.

Especially if the volume 33 of the wells 11 is small, the wells 11should be sealed against the outside atmosphere. For this purpose, acoverplate shown in FIG. 4 is provided which consists of a thin coverfilm 49. Through-holes 50, which are arranged in the same grid patternas the through-holes 9 in the plate 2, are provided in the cover film49. The cover film 49 has a plane upper side 51 and an underside 52which is parallel thereto and with which the film comes to lie on theupper side of the plate 2 when the latter is covered. The cover film 49has a thickness 53, measured between the upper side 51 and the underside52, which is small compared with the transverse dimensions of the coverfilm 49. The cover film 49 is, for example, made of a polycarbonatehaving a thickness of 0.1 mm.

When it is placed on the plate 2, the cover film 49 is aligned such thatthe through-holes 50 are aligned with the through-holes 9. In this way,the cover film 49 and the plate 2 can simultaneously be joined to oneanother and be fixed to a support device, in a manner which will bedescribed in more detail.

Of course, instead of the through-holes 50 or the through-holes 9, it ispossible to provide cylindrical pegs which project downwards or,respectively, upwards and which engage in the through-holes 9 or,respectively, the through-holes 50 when the cover film 49 is placed onthe plate 2 and thus join the cover film 49 to the plate 2 in adetachable manner.

The material preferably used for the cover film 49 is, as alreadymentioned, a polycarbonate having a thickness of 0.1 mm. This film istransparent in the wavelength range of interest for the opticalanalytical methods used and displays only slight inherent fluorescence.The optical analytical methods can thus also be applied from abovethrough the cover film 49; in particular it is possible, using theirradiation method through the cover film 49 and the bottom wall 24 ofthe wells, to measure the optical density of the substances placed inthe wells 11.

In the case of the preferred small volumes 33 of the wells 11, which arein the range between 30 and 100 μl, the volume of solutions placed inthe wells 11 can change as a result of condensation and/or evaporationeffects. This applies in particular if frequent changing of thetemperature of the solutions between high and low temperatures isrequired, as occurs in the case of the polymerase chain reaction (PCR),a method frequently used for high amplification of individual nucleicacid strands.

In order to increase the sealing effect of the cover film 49, the coverfilm 49 is joined to the plate 22 in the region of each well 11 by aclosed circular joining seam 55 surrounding the opening edge 17 of thewell. In FIG. 5 it can be seen that each joining seam 55 bounds acircular area 57 of the cover film 49, which in each case covers theopening 18 of an assigned well 11. In this way each well 11 is so to saycovered by its own lid in the form of the circular area 57, which, bymeans of the joining seam 55 is joined in such a way with the bridges 29surrounding the well 11 that each well 11 is sealed gas-tight againstthe atmosphere and the other wells 11.

A welding die 59 which is profiled on its front face 58 and is shown insection in FIG. 6 serves, for example, for making the individual joiningseams 55. The welding die 59 has a solid cylindrical base body 60, whichat its upper end 61 carries an annular attachment 62 which is integralwith the base body 60. The annular attachment 62 delimits a circularrecess 63, which is concentric with the base body 60 and thus with itslongitudinal axis 64, and carries the ring-shaped front face 58 facingaway from the base body 60.

Profiling in the form of pyramids 65 arranged in rows, which pyramidsare constructed in one piece with the annular attachment 62 at theirsquare base 66, is provided on the front face 58 which surrounds therecess 63 in ring form. The tips 67 of the pyramids 65 face away fromthe base body 60 in a direction parallel to the longitudinal axis 64 ofthe welding die 59.

FIG. 7 shows the top view on the front face 58 in the direction of thearrow VII from FIG. 6 in section. As can be seen, the pyramids 65 arearranged in rows 68 and 69 which are offset relative to one another byhalf the width of the pyramid base 66. The arrangement is such that arow 69/1 runs between two rows 68/1 and 68/2, which are parallel to oneanother and are not offset relative to one another, which row 69/1 isaccordingly offset relative to the rows 68/1 and 68/2 by half the widthof the pyramid base 66. The row 68/2 is directly followed, remote fromthe row 69/1, by a row 69/2, which is parallel to the row 69/1 andaligned laterally with respect to the latter.

Returning to FIG. 6 it can be seen that the welding die 59 is providedwith a heater, indicated diagrammatically at 71, by means of which thewelding die 59, which is preferably made of V2A steel, is heated toabout 280° C. In order to make the joining seam 55, the heated weldingdie 59 is placed from above onto the upper side 51 of the cover film 49,which is laid on the plate 2, in such a way that its profiled,ring-shaped front face 58 centrally surrounds the edge 17 of the openingof the well 11 which is located below the cover film 49 and is to besealed. The circular recess 63 has a diameter which is so large that thetips 67 of the pyramids come to lie outside the edge 17 of the openingon sections of the cover film 49 which are located above the bridges 29.

The square base 66 of the pyramids 65 measures 0.5×0.5 mm and the tip 67of the four-sided pyramid 65 lies 0.25 mm vertically above the pyramidbase 66, i.e. two sides of a pyramid which are opposite one anotherenclose a vertical and opposite angle of 90°. In the radial direction upto three pyramids 65 are arranged in succession on the ring-shaped frontface 58, so that the welding die 59 overall has an external diameterwhich is larger by at least 6 base lengths of a pyramid 65 than thediameter of the recess 63.

The following procedure has proved suitable for welding a cover film 49,the thickness 53 of which is about 0.1 mm, to a plate 2, the thickness 7of which is about 0.27 mm:

The cover film 49 is placed from above onto the plate 2 in such a waythat it covers the wells 11 and that the through-holes 50 are alignedwith the through-holes 9. The welding die 59, which is heated to 280°C., is placed from above, with its front face 58 first, onto the upperside 51 of the cover film 49 in such a way that it is located centrallyabove a well 11 which is located below the cover film 49 and is to bewelded. The pyramids 65 on the front face 58 now lie with their tips 67,which may penetrate somewhat into the material of the cover film 49, onthe upper side 51 and heat the latter. The cover film 49 is pre-heatedin this way for about 13 seconds by the honeycomb profile of the frontface 58. The welding die 59 is then pressed downwards through about0.1-0.2 mm onto the cover film 49, so that each pyramid 67 penetratesinto the cover film 49 and this in turn penetrates into the bridges 29of the plate 2. The welding die 59 remains in this position for twoseconds and is then removed completely from the cover film 49.

The joining seam 55 which is thus produced and which is a type of weldseam is shown in FIG. 8 in a cross-section along the line VIII--VIIIfrom FIG. 5. The cooled down joining seam 55 has a profilingcorresponding to that of the welding die 59. The pyramids 65 havepressed into the preheated upper side 51 of the cover film 49upside-down pyramid-like depressions 73, which in respect of their shapecorrespond to the pyramids 65. In addition, in the region of thedepressions 73, the cover film 49 has penetrated with its underside 52into the upper side 5 of the bridges 29, which is indirectly preheatedthrough the cover film 49, and has formed depressions 74, whichcorrespond to the depressions 73, in said upper side 5. In this way acontact surface 75, which in cross-section has a zig-zag form, hasformed between the underside 52 of the cover film 49 and the upper side5 of the plate 2. As a result of this zig-zag shape, the contact surface75 is larger than the bearings surface which existed before the weldingbetween the underside 52 of the cover film 49 and the upper side 5 ofthe plate 2 in the envisaged region of the joining seam 55.

Not only has the bearing surface been enlarged by the heating action ofthe pyramids 65; the cover film 49 and the bridges 29 have also beenintegrally welded to one another along the contact surface 75. It hasbeen found that this joining seam 55 ensures a good, not onlyliquid-tight but also gas-tight, seal of the individual wells 11 even inthe case of frequent changing between high and low temperatures at theunderside 6 or the outside 28. This even holds true in case a pressureabove atmospheric arises inside of the wells due to the fact that thesolutions placed in the wells are heated or carried to such a hightemperature that the gas volume above the solution tends to expand. Thejoining seam 55 also withstands, without any problems, the customarymechanical stresses to which the sealed plate 2 is subjected in everydaylaboratory practice, as well as the slight changes in shape and strainswhich are associated with changing the temperature.

During the welding operation described above, the circular section 57 ofthe cover film 49 which covers the opening 18 bulges upwards in adome-like manner, so that a plate 2 which is sealed and welded asdescribed above has a lens-like bulge 76 in the cover film 49 above eachwell 11.

Since, however, the bulge 76 is formed only in the case of wells 11which are welded gas-tight, it is at the same time an optical indicationthat the joining seam 55 formed has provided a gas-tight seal of therelevant well 11, which seal is also capable of withstanding to anoverpressure having arisen inside of the wells. If the cover film 49does not display any bulges 76 after welding then, for example, thewelding operation was defective in respect of the residence times, thetemperature of the welding die 59 or the depth of penetration of thepyramids 65 into the upper side 51.

In the illustrative embodiment described, the temperature of the weldingdie 59, the dimensions of the pyramids 65 and the depth of penetrationof the pyramids 65 into the upper side 51 of the cover film 49 areindicated merely by way of example for a cover film made ofpolycarbonate having a thickness of 0.27 mm. For thicker polycarbonatefilms, the depth of penetration of the pyramids, which approximatelycorresponds to the thickness of the cover film, must be adjusted to thenew thicknesses.

In addition to correctly maintaining the residence time of the weldingdie 59, initially on the upper side 51 and then in the position where ithas penetrated into the upper side, the depth to which the pyramids 65penetrate into the material of the cover film 49 is also important forthe success of the welding operation. Although the welding operationdecsribed above can be carried out manually, the yield of correctlypositioned joining seam 55 is substantially increased by the use of awelding installation 78 as shown in FIG. 9.

The welding installation 78 has a flat, rectangular baseplate 79 and aflat top plate 80, which is arranged above the baseplate 79 and hasapproximately the same transverse dimensions as the baseplate 79. Thetop plate 80 is fixed to the baseplate 79 with the aid of four guiderods 81. Of the four guide rods 81, which are screwed into the baseplate79 from above, each in the region of one of the four corners the frontrighthand guide rod 81/4 has been omitted from FIG. 9 for reasons ofclarity in the drawing.

A support plate 82, the height of which is adjustable and in the outercorners of which spherical liners 83 are let in, through which the guiderods 81 pass, is provided between the baseplate 79 and the top plate 80.As drive for the height adjustment of the baseplate 82, an electricallyoperated drive motor 84 is attached, by its flange 85 from above on thetop plate 80, remote from the baseplate 82. The motor 84 has a motorshaft, indicated at 86, which is connected to a ballscrew pinion gearindicated at 87. The ballscrew pinion gear 87 is connected on the otherside to the support plate 82 and serves to translate the rotary movementof the motor shaft 86 into the adjusting movement of the support plate82 along the guide rods 81.

Remote from the ballscrew pinion gear 87, a heating block 89, which isfixed from below to the support plate 82 by four distance bolts 90, isprovided centrally below the support plate 82. The heating block 89,which is made of copper, fulfills the role of the heater indicated at 71in FIG. 6 for the welding die 59, three of which are indicated in FIG.9. The welding dies 59/1, 59/2 and 59/3 plug, remote from the distancebolts 90, from below into the heating block 89 and their front faces 58face downwards away from the heating block 89.

A blind hole 91, in which an electrically heatable heating cartridge isinserted, which for reasons of clarity is not shown in more detail, isprovided in the heating block 89 and in FIG. 9 passes virtuallycompletely through said heating block from right to left. Thetemperature of the heating block 89 is suitably measured by atemperature probe, which is not shown in more detail, and passed to acontrol circuit, which is likewise not shown and which, in turn,controls the heating cartridge. A closed control circuit, via which thetemperature of the heating block 89 is kept at a constant value, forexample 280° C., is thus formed in a manner known per se. Via thedistance bolts 90, the heating block 89 heats the support plate 82,which can result in jamming of the spherical liners 83 on the guide rods81. For this reason, coolant bores 92, through which the support plate82 is connected to a thermostat-controlled cooling circuit, are providedin the support plate 82. In this way, the temperature of the supportplate 82 can be adjusted independently of the temperature of the heatingblock 89 via an external thermostat, so that a smooth-running adjustmentof the support plate 82 along the guide rods 81 is ensured.

A receiving block 93, which faces upwards, is provided on the baseplate79 approximately centrally below the heating block 89, the height ofwhich can be adjusted via the support plate 82. The receiving block 93has a coolant bore 94 which passes through it and which is connected, inthe same way as the coolant bore 92 of the support plate 82, to anexternal thermostat circuit which keeps the receiving block 93 at aconstant and adjustable temperature.

The receiving block 93 has cups 95 which are open at the top and aredesigned to receive the protuberances 21 projecting downwards beyond theplate 2. The cups 95 therefore have the same dimensions as the blindholes 38 which can be seen in FIG. 3 in the shaping block 37 and, likethe wells 11, are arranged in rows 12 and columns 13.

A plate 2 is placed from above on the receiving block 93, which plate 2is in turn covered by a cover film 49. A perforated mask 96, whichengages around the receiving block 93 on all sides from above, isslipped over the cover film 49 and presses the cover film 49 onto theplate 2 and the latter, in turn, with its wells 11 into the receivingblock 93. Through-holes 97, aligned with the welding die 59, areprovided in the perforated mask 96, said holes likewise being arrangedin rows 12 and columns 13 in such a way that one hole 97 is centrallyaligned above each well 11. For reasons of clarity, the perforated mask96, the cover film 49 and the plate 2 are shown broken off and offsetrelative to the receiving block 93.

Of course, a hole 97 and a welding die 59 are provided for each well 11in the plate 2.

On either side of the receiving block 93 two identical upwardly facingpedestals 98 are arranged for fixing the perforated mask 96 on thebaseplate 79, the righthand pedestal 98/2 of which is shown broken off.The pedestal 98/1 has a fixing bore 99, which faces upwards and to whicha fixing clamp, which, for example, can be constructed as a spring clampor as a locking bar, is fastened in order to press the perforated mask96 downwards onto the receiving block 93.

For reasons of clarity, the fixing clamp has been omitted in FIG. 9.

The welding installation 78 which has been described thus far operatesas follows:

The support plate 82 is in the raised starting position shown in FIG. 9.After the perforated mask 96 has been removed from the receiving block93, a plate 2, to be welded, is placed from above on to the receivingblock 93 in such a way that the wells 11 come to lie with theirprotuberances 21 in the cups 95. The wells 11, the openings 18 of whichface upwards, are already filled with the desired substances and coveredby a cover film 49, or are now appropriately filled and then coveredwith a cover film 49, which is aligned such that its through-holes 50are aligned with the through-holes 9 in the plate 2. The perforated mask96 is slipped over the plate 2 covered in this way, the through-holes 97of said mask coming to lie centrally above the wells 11. With the aid ofthe fixing clamps provided on the pedestals 98, the perforated mask 96is pressed firmly downwards onto the receiving block 93.

The heating block 89 is heated to 280° C. by means of the heatingcartridge inserted in the blind hole 91. The welding dies 59, which areconnected to the heating block 89 in a thermally conducting manner, alsohave this temperature. Via the ballscrew pinion gear 87, the rotarymovement of the motor shaft 86 of the drive motor 84 is translated intoa downwards movement of the support plate 82 guided via the sphericalliners 83 and the guide rods 81. As the support plate 82 and thus theheating block 89 are lowered, the welding dies 59/1 and 59/2 push fromabove into the assigned holes 97/1 and 97/2 respectively in theperforated mask 96. The transmission of the ballscrew pinion gear 87 andthe number of revolutions of the motor shaft 86 are set such that at theend of the downwards movement of the support plate 82 the welding dies59 come to lie with their front face 58 or the tips 67 of the pyramids65 just in contact with the upper side 51 of the cover film 49, as hasalready been described above.

The welding installation 78 pauses for about 13 seconds in thisposition, in which the welding dies 59 preheat the cover film 49 and theplate 2 in the area where the joining seams 55 are to be made. Afterthis preheating time, the support plate 82 is gradually moved 0.1 mmfurther downwards towards the receiving block 93, via the ballscrewpinion gear 87 of the motor 84, so that the pyramids 65 on the frontface 58 of the welding dies 59 penetrate into the cover film 49 and thelatter penetrates into the bridges 29 of the plate 2. After a furthertwo seconds, the motor 84 is driven such that its motor shaft 86 turnsin the direction opposite to the previous direction of rotation and, viathe ballscrew pinion gear 87, the support plate 82 and thus the heatingblock 89 and the welding dies 59 are thus raised again to the initialposition shown in FIG. 9.

After loosening the fixing clamps, the perforated mask 96 can be removedand the plate 2, which is welded as shown in FIG. 5, is removed from thereceiving block 93. The next plate 2 is now placed on the receivingblock 93 and the welding operation begins anew.

For many experiments it is necessary to keep the substances placed inthe wells 11 at low temperatures to prevent their being heated duringthe welding operation just described. For this purpose, the receivingblock 93, and thus its cups 95, is thermostat-controlled via the coolantbore 94 to a temperature which is required for the particularsubstances, for example to 10° C. The wells 11 lie with their heatexchange surface 28' close against the inner wall of the particular cup95, so that, because of the low thickness 31 of the wall 20 of the wells11, the substances present in the wells 11 are kept at the sametemperature as the receiving block 93 itself. Because of the good heattransfer, the heat which may be supplied to the substances duringwelding is instantaneously dissipated through the wall 20 into thereceiving block 93.

In this way, substances which have a very sensitive reaction totemperature variations can also be welded in the wells 11 of the newplate 2 with the aid of the novel welding installation 78. Consequentlyit is possible, to a degree not known hitherto, to packagetemperature-sensitive substances or solutions, or highly infectioussubstances, gas-tight in a large number in an extremely small space. Thesubstances can be, for example, reaction solutions prepared forbiochemical and/or microbiological test methods, which are supplied tothe user already in portioned and welded form in the novel plates 2. Thesubstances to be tested by the user can, for example, be introduced intothe test solutions present in the wells 11 by piercing the bulges 96,covering the openings 18 of the wells 11, from above using a thin hollowneedle. The substances to be tested are then injected into the testsolutions present in the wells 11.

After withdrawing the hollow needle, which, for example, is a syringecommonly used in everyday laboratory practice, a capillary-like channelremains in the bulge 76. Exchange of moisture with the surroundingatmosphere is not possible via this channel, so that the volume of thesubstances or solutions placed in the wells 11, which have been weldedgas-tight, does not change as a result of condensation or evaporationeffects.

Usually, however, the wells 11 of the novel plate 2 are filled in situ,for example in the chemical laboratory, and sealed gas-tight with acover film 49 using the novel welding installation. The fixed gridpattern of the columns 13 and rows 12 makes it possible, with thisprocedure, to fill several wells 11 at the same time using a multiplepipette known per se.

FIG. 10 shows a plate 2 with wells 11, sealed gas-tight, in which, forexample, solutions are present, the course of reaction of which can beinfluenced via their temperature. The solutions have either been filledinto the wells 11 in situ or were already in the plate 2, supplied as awelded plate, and were subsequently inoculated by the user with thesubstances to be tested--for example DNA molecules to be tested.

The plate 2 prepared in this way is inserted from above into athermoblock 101 which has blind holes 102 which are open at the top andserve to receive the beaker-like protuberances 21. The blind holes 102have the same shape as the blind holes 38 in the shaping block 37 usedto produce the plate 2. After the protuberances 21 have been inserted inthe blind holes 102, the inner wall 103 of said blind holes liesdirectly against the heat transfer surface 28' of the protuberances 21.Therefore there are no layers of air between the outside 28 and theinner wall 103, which acts as counter-surface 103', interfering with theheat transfer between the thermoblock 101 and the interior 19 of thewells 11.

Threaded holes 104, which are open towards the top, are also provided inthe thermoblock and, when the plate 2 is inserted in the thermoblock101, align with the through-holes 50 and 9 in the cover film 49 and inthe plate 2, respectively. Screws 105 are screwed into the threadedholes 104 from above through the through-holes 50 and 9, and the plate 2sealed with the cover film 49 is thus firmly connected to thethermoblock 101. The upper side 106 of the thermoblock 101 comes to lietightly against the underside 6 of the plate 2 and the protuberances 21are pressed firmly with their heat exchange surface 28' on the innerwall 103 of the blind holes 102.

Because of the smooth surface of the outside 28, which is in directthermal contact with the inner wall 103, and because of the high heattransmission coefficient described, the solutions present in the wells11 assume the temperature of the thermoblock 101 within a few seconds.If the solutions are, for example, to be stored for a prolonged periodat a low temperature, the thermoblock 101, which is made of a metalhaving good thermal conductivity, is temperature-controlled, for exampleto +4° C., via a thermostat connected to it.

If the reaction in the solutions is to be initiated, the thermoblock 101is heated in a suitable manner to the reaction temperature of thesolutions, which, because of the good heat transfer, follow thetemperature change in the thermoblock 101 virtually immediately. Thetemperature change in the thermoblock 101 itself can be effected in amanner known per se by immersing the thermoblock 101 in water baths ofdifferent temperatures, by bringing the thermoblock 101 intoheat-conducting contact with further metal blocks pre-regulated to thedesired temperature, or by moving the thermoblock 101 along a metal railon which a temperature gradient has been established.

In particular the metal rail with the temperature gradient makespossible the cyclic changing of the temperature of the thermoblock 101and thus of the temperature of the solutions in the wells 11. Forcarrying out the polymerase chain reaction in the wells 11, thethermoblock 101 is, for example, first kept at 37° C. for 60 seconds,then at 72° C. for 120 seconds, then at 94° C. for 60 seconds and thenagain at 37° C. for 60 seconds, etc. Due to the even at overpressuregas-tight seal of the single wells, even at the high temperatures nowater vapour saturated gas can escape from inside the wells. The amountof water vapour in the air volume above the solution placed in the wellis controlled by the fluid, however, since no air can escape, this willnot lead to any evaporation processes, so that the initial concentrationof the solutions will not vary during the great number of temperaturecycles. This provides for a good yield of the experiment.

The time which is needed to bring the solutions to the individualtemperatures is decisive for the course of the polymerase chainreaction. Whereas a typical course of reaction in the known plasticreaction vessels takes more than 10 hours and is usually carried outovernight, when the new plate 2 is used the reaction is complete in lessthan 4 hours. An experiment of this type can, therefore, now beprepared, carried out and analyzed within one day.

After the experiment has proceeded to completion the solutions are atleast partially further used, for example to analyze them on aseparating gel. For this purpose, the bulge 76 is punctured using thesyringe indicated at 107 in FIG. 10 and a portion of the solution isremoved. After withdrawing the syringe 107, the solution remaining inthe well 11 can be stored, for example in the manner described above.Although the hole formed in the bulge 76 during sampling does not resultin any significant exchange of moisture, it can subsequently be sealedagain, for example using an adhesive film.

Finally, it may be mentioned, solely for the sake of completeness, thatthe transverse dimensions of the new plate 2, and also the row andcolumn spacings 14 and 15 respectively, essentially depend on the fillvolume 33 of the wells 11 which is desired in the particular case. Thethermoblocks 101, receiving blocks 93 and shaping blocks 37 used in eachcase are adapted to these spacings. In every case, however, thethickness of the film 36 is so chosen that the wells 11 in the finishedplate 2 have a bottom wall 24 which has a thickness 31 in the region of0.04 mm, so that the heat transfer coefficient has the necessary highvalue.

We claim:
 1. Process for producing a plate member comprising at leastone well having its opening facing upwards, for receiving chemicaland/or biochemical and/or microbiological substances, said wells beingmade in the plate member with the aid of a molding die means, whereinthe process comprises the steps of:a) applying a plastic sheet ofsynthetic material, which sheet is deformable by the action of heat, ona molding block means which serves as said molding die means and whichhas recessed portions with the negative shape of the wells to be formed;and b) subjecting said plastic sheet, for a predetermined period oftime, to the action of a hot gas flow of a predetermined firsttemperature, the gas flow impinging upon at least an area of saidplastic sheet covering one of the recessed portions, heating up thisarea and pressing it into said recessed portion so that it comes to lieflat against the latter's inner wall which is smooth throughout, and c)maintaining said molding block means at a predetermined secondtemperature which is lower than said predetermined first temperature,but above room temperature,wherein said plastic sheet is a polycarbonatefilm having a thickness smaller than 0.5 mm, said predetermined firsttemperature is between 250° C. and 300° C., and said predeterminedsecond temperature is between 90° C. and 110° C.
 2. Process forproducing a plate member comprising at least one well having its openingfacing upwards, for receiving chemical and/or biochemical and/ormicrobiological substances, said wells being made in the plate memberwith the aid of a molding die means, wherein the process comprises thesteps of:a) applying a plastic sheet of synthetic material, which sheetis deformable by the action of heat, on a molding block means whichserves as said molding die means and which has recessed portions withthe negative shape of the wells to be formed; and b) subjecting saidplastic sheet, for a predetermined period of time, to the action of ahot gas flow of a predetermined first temperature, the gas flowimpinging upon at least an area of said plastic sheet covering one ofsaid recessed portions, heating up this area and pressing it into saidrecessed portion so that it comes to lie flat against the latter's innerwall which is smooth throughout, and c) maintaining said molding blockmeans at a predetermined second temperature which is lower than saidpredetermined first temperature, but above room temperature, suchthatthe at least one well is formed with a wall having a smooth anduniform outer surface and a defined contour, which contour can bebrought into direct heat-exchanging contact with a corresponding shapedcounter-surface, enabling a rapid heat-exchange through said wall ofsaid well and thus providing for a rapid temperature change of saidsubstances, and wherein said plastic sheet is a polycarbonate filmhaving a thickness smaller than 0.5 mm, said predetermined firsttemperature is between 250° C. and 30020 C., and said predeterminedsecond temperature is between 90° C. and 110° C.
 3. Process forproducing a plate member comprising at least one well having its openingfacing upwards, for receiving chemical and/or biochemical and/ormicrobiological substances, said wells being made in the plate memberwith the aid of a molding die means, wherein the process comprises thesteps of:a) applying a plastic sheet of synthetic material, which sheetis deformable by the action of heat, on a molding block means whichserves as said molding die means and which has recessed portions withthe negative shape of the wells to be formed; and b) subjecting saidplastic sheet, for a predetermined period of time, to the action of ahot gas flow of a predetermined first temperature, the gas flowimpinging upon at least an area of said plastic sheet covering one ofsaid recessed portions, heating up this area and pressing it into saidrecessed portion so that it comes to lie flat against the latter's innerwall which is smooth throughout, and c) maintaining said molding blockmeans at a predetermined second temperature which is lower than saidpredetermined first temperature, but above room temperature, suchthatthe at least one well is formed with a wall having a smooth anduniform outer surface and a defined contour, which contour can bebrought into direct heat-exchanging contact with a corresponding shapedcounter-surface, enabling a rapid heat-exchange through said wall ofsaid well and thus providing for a rapid temperature change of saidsubstances, and wherein said hot gas flow issues from a nozzle meanshaving an orifice, which is maintained at a fixed distance from saidplastic sheet during said predetermined period of time, and said welldisplays a circular cross-section, has a volume smaller than 200 mm³ anda diameter smaller than 10 mm, said orifice of said nozzle means has adiameter approximately equal to the diameter of said well and said fixeddistance corresponds approximately to said diameter of said well.
 4. Aprocess as claimed in 3, wherein said plastic sheet is a polycarbonatefilm having thickness smaller than 0.5 mm, said predetermined firsttemperature is between 250° C. and 300° C., and said predeterminedsecond temperature is between 90° C. and 110° C.